Research of Cryotherapy

(1. School of Medicine Tsinghua University, Beijing 100084, China; 2. Department of Dermatology, the First Affiliated Hospital of Dalian Medical University, Dalian 116011, China)

Abstract: Cryotherapy is a kind of physical treatment that utilizes freezing temperature to induce local tissue ablation and destroys certain live tissue controllably, which has most therapeutic applications in dermatology and oncology. Handheld nitrogen spraying equipment is mainly used in treating skin lesions while automatic multiple-probe freezing systems such as argon-helium knife and combined knife are used in treating a variety of solid tumors. Recently, researches have covered most of the mechanisms, techniques and clinical analysis of cryotherapy. However, genome-level variations in immune response to cryotherapy and combined therapy with immunological drugs still warrants further investigation to reveal the nature of cryotherapy and enhance its clinical effect.

Key words: Cryotherapy; Tumor ablation; Freeze-thaw cycle; Endocare CryocareTM Surgical System; HYGEATM Combined Knife

In recent years, cryotherapy has been used for the treatment of various skin lesions and has become an important means of treating solid tumors that cannot be removed by surgery. As a new local treatment, cryotherapy has the advantages of effective and accurate ablation, compared with other treatments such as radiofrequency ablation and hyperthermia. It can destroy pathological cells directly by causing cell necrosis and apoptosis, changing the local microcirculation and regulating the immune system to remove the skin lesions and malignant tumors. The use of cryotherapy has less adverse reactions and better tissue repair effect [1]. With the development of cryotherapy device, the improvement of cryoablation technology and the accumulation of clinical experience, cryotherapy plays an increasingly important role in the treatment of skin diseases and tumors. The field of application of cryotherapy is constantly expanding. This paper describes the development of cryotherapy device and the current situation of refrigerant use, compares the cryotherapy principle and characteristics of argon-helium knife widely used at present and Combo knife newly marketed, and reviews the research of cryotherapy at home and abroad, with a view to promoting the improvement of the therapeutic effect of the cryotherapy technique and the expansion of the field of treatment.

1 Cryotherapy device and refrigerants

1.1 Development of cryotherapy device. Currently, hand-held liquid nitrogen spray devices consisting of a refrigerated storage tank and a nozzle are primarily used clinically to perform spray cryosurgery for skin diseases. With the widespread use of cryosurgery, automated multi-probe cryosurgery device has been developed. In 1998, Endocare Corporation of the United States successfully developed a tumor treatment device, the U.S. argon-helium knife. Under the guidance of CT or B-ultrasound, the argon-helium knife head can be accurately penetrated into the center of tumor tissue percutaneously, when the tumor tissue can be reduced to -100°C by rapid adiabatic refrigeration of argon at the knife tip; the ice ball can then be thawed and rapidly heated to 50°C by rapid heating of helium on the tip.[2] Prof. Liu Jing's team, in conjunction with HYGEA, has independently developed a large-scale tumor treatment device - Combo knife. The Combo knife treatment system can reach temperatures as low as -196°C and as high as 80°C or more [3]. Both Combo knife and argon-helium knife are designed to kill cancer cells by switching rapidly between high-intensity refrigeration and heat production, but the Combo knife has significantly better overall performance than that of the argon-helium knife, and lower cost of both the probe and the liquid nitrogen [4]. A variety of different types of cryotherapy device are now available for clinical use.

1.2 Refrigerants. Although liquid carbon dioxide, nitrous oxide, freon, or argon can also be used as refrigerants in clinical treatment, liquid nitrogen is still the most commonly used modern refrigerant. Liquid argon is used to cool the probe tip at 300 atmospheres by the Joule-Thomson (J-T) effect, which means that the gas is transferred by the pressure of 1 restriction hole in the heat exchange chamber of the probe. In the J-T apparatus, liquid argon offers faster cooling than liquid nitrogen, but only at -130 to -135°C, and the gas reflux after freezing rapidly heats the probe. In the treatment of tumors, due to the lower freezing capacity of liquid argon, liquid argon requires twice as many probes to achieve the same freezing effect compared to the freezing diameter of liquid nitrogen. However, liquid nitrogen vacuum insulation storage tanks are not required for the J-T apparatus with pressurized argon gas for refrigeration. The argon-helium knife belongs to the J-T apparatus, and the argon gas working at 300 to 500 atmospheres is a rare ultra-high-pressure gas, with restricted access and safety hazards in the surgical implementation; whereas, the Combo knife works by freezing by change of state, using liquid nitrogen with an operating pressure of 2 to 5 atmospheres, which is easy to access and has good safety and operability [5]. The other two cryogens commonly used in the J-T apparatus are liquid carbon dioxide and liquid nitrous oxide, which, although used clinically for many years, lack sufficient freezing capacity to treat aggressive cancers or the freezing capacity as multi-probe devices.

2 Cryotherapy techniques

2.1 Cryotherapy methods. There are three main methods: application by a cotton swab, targeted spraying and probe intervention. The first two methods are mainly used for the treatment of various skin diseases, while the probe intervention is mainly used for the treatment of various tumors. Since the virus may survive in liquid nitrogen and contaminate the refrigerant, the method of application by a cotton swab requires separate distribution of refrigerant for each patient to avoid cross-infection [6]. In addition, the maximum freezing depth of liquid nitrogen application by a cotton swab is 2 - 3 mm, so it cannot be used for the treatment of deep lesions. Targeted spraying is a spray treatment using a hand-held liquid nitrogen spray unit, with three main modes: direct mode (spraying the center of the lesion directly), spiral mode (spraying from the center of the lesion in a spiral outward), and "paintbrush" mode (moving the nozzle from one side to the other lesions in a manner of painting a wall). The use of handheld thermal insulation plastic cone is an improvement of the targeted spraying treatment. The cone with appropriate size is directly placed in the lesion area, so that the spray area of the refrigerant is limited to the area exposed seeing from the cone tip, deepening the depth of freezing compared to the use of sprays alone, and the refrigerant is applied directly to the target tissues, which allows for the therapeutic effect of deep freezing and extensive freezing with the slower cooling rate of the refrigerant [7]. The cone tip has different sizes and shapes, which allow the formation of a variety of frozen blocks in the target tissue. Probe intervention is a therapeutic approach to cryosurgery using probes, where one or more probes are inserted into the deeper tissues of the tumor, and the refrigerant is recycled through the needle under pressure and turbulence, resulting in the formation of an ice ball within the deeper tissues of the tumor being treated. 2 freeze-thaw cycles can ablate up to 85% of solid tumors, and multiple freezes are required for tumors that are deeper or larger in diameter. Combo knife and argon-helium knife are devices for probe intervention.

2.2 Cooling rate. In cryotherapy, rapid freezing (cooling rate >50°C/min) occurs only in the vicinity of the cryoprobe, with lower cooling rates the further away from the probe. About 1cm from the center of the spray, the cooling rate is only 10~20°C/min, and is related to the nozzle temperature. Most tissues are not rapidly cooled when cryotherapy is performed on tumors > 2 cm in diameter, but low cooling rates do not seem to alter the treatment outcome [8]. Farrant and Walter[9] suggest that the main factor determining cell survival is the different heat changes that cells are exposed to at different times, not the cooling rate. The time taken to freeze different lesion tissues also varies considerably.

2.3 Temperature control. The small volume of intracellular water and the diversity of electrolytes lead to different freezing points, even as low as -54.9°C (the lowest freezing point of calcium chloride solutions). In tumor therapy or other situations where tissue destruction must be ensured by cryogenics, -50°C is safest for all tumor tissues, considering the margin of error for clinical applications. There is no requirement for freezing duration if the tissue is kept below -50°C, whereas a frozen state (e.g., above -40°C) will increase tissue destruction. When the tissue temperature is higher than -40°C, the longer the thawing time, the more obvious the solute effect and ice crystal growth, and the stronger the damage to cells [8].

2.4 Freeze-thaw cycle. Repeated freeze-thaw cycles are more destructive to tumor tissue, but the specific intervals between freeze-thaw cycles have not been determined at this time. Whittaker[10] found in cyclic repetitive freezing experiments on hamster oral mucosa that intracellular ice crystals increased in the interval between each freeze-thaw cycle, leading to the proposal that longer intervals increase the effectiveness of freezing, and that longer thawing times induce osmotic disruption of cell membranes by re-crystallization of intracellular ice crystals. At the same time, continuous thawing will keep the tissue in a hypothermia state that lasts long enough to stagnate microcirculation. When heat loss occurs in this frozen region, the 2nd freeze-thaw cycle will be more effective. However, in clinical practice, it is difficult to wait for long intervals for the ice crystals to completely thaw due to the workload. In summary, the most desirable technique for destroying pathological tissue is to rapidly freeze the tissue to an appropriate low temperature, extend the duration by slowly thawing, and repeat the freeze-thaw cycle.

3 Simple mechanisms of cryotherapy

3.1 Superficial phenomena. When cryospray is applied to skin lesions, an apparent change occurs immediately after a freezing period of 20 - 30s, displaying a white ice field. Within a few minutes of thawing, the skin at the edge of the ice field appears purple-red and moves in a concentrated manner, with distinct boundaries from the surrounding healthy skin in both the epidermal and deeper layers of the skin, followed by the deeper tissues becoming paler, and hemorrhagic blisters formed on the surface gradually forming scabs for a period of 2 to 6 weeks [11]. Essentially, there is no difference in the changes after cryo-freezing and cryo-spray treatment; ice crystals can be seen immediately upon freezing, but the 1st cellular changes are delayed until after about 30 min when increasing numbers of eosinophils appear in the cytoplasm; within 1 h after freezing, there may be no obvious cell death, but karyopyknosis and homogenization of the cytoplasm can occur in the following hours [12].

3.2 Cell necrosis and apoptosis. Cell necrosis or gene-regulated cell death (apoptosis) is a mechanism by which freezing directly results in cell damage. A cryopreservation study of in vivo experiment conducted by Forest et al [13] in which a human lung adenocarcinoma cell was injected into the dorsal region of mice showed partial necrosis in the center of the frozen region with apoptotic cells in the surrounding region, and apoptosis gradually increased in the period of 2-8 h after freezing, which took place in the region beyond the necrotic region, but the boundary between the two was not clear, and the 2nd necrosis was observed after 4d. The in vivo experiment illustrates the timing of apoptosis after cryosurgery and also clarifies the direct link between in vitro and in vivo studies. Apoptosis or secondary necrosis is caused by the changes of microenvironment inside and outside tumor cells during freezing, such as pH value change, ATPase abnormality, mitochondrial damage, electrolyte disturbance, organelle and nucleus rupture.[14].

3.3 Direct cell damage. Frozen site microscopically shows endoplasmic reticulum degranulation. Histologically, blisters appear at the dermal-epidermal junction, and maximal death of epidermal and associated melanocytes occurs. Cell membrane disruption due to extracellular ice crystals is disruptive to tightly packed cells in solid tumors. During freezing, ice crystals form in many cells, destroying mitochondria and endoplasmic reticulum. Changes in extracellular ice and decreases in extracellular water are associated with increases in solute concentration. Changes in osmotic pressure can lead to cell lysis, cell volume reduction, and cell membrane rupture, some of which are irreversible and exacerbated during thawing. Although many of the cells look normal right after thawing, many of them have died. Large ice crystals are more destructive than small ice crystals [12]. Slow thawing is associated with the recrystallization of ice and is more destructive than rapid thawing. Liquid nitrogen cryosurgery is most effective in clinical practice because of the slow thawing of liquid nitrogen relative to other refrigerants, which is an important factor in increasing tissue destruction.

3.4 Microcirculatory arrest and regeneration. Gentle cooling reduces microvascular flow, which is easily observed in winter when the skin turns white, but this effect is less in arterioles. After liquid nitrogen freezing, blood vessels contract, but after deep freezing, blood vessels begin to expand, microthrombi form in capillaries and veins and gradually become fixed to the endothelial cells, so that they eventually do not flow at all, and this effect can be observed below -15°C. Microthrombi are formed due to platelet aggregation and blood stasis, resulting in cell avascular necrosis, and the degree of cell avascular necrosis will depend on freezing depth and lateral diffusion width [15]. In clinical practice, occlusion of veins and capillaries can occur, but rarely of large arteries and arterioles.

The tissue is frozen, but the matrix may not change much, and the preservation of this architecture is important for repair. Wound healing is an active process that begins with an inflammatory response at the border of the lesion due to chemokines, with an early onset of neutrophil infiltration, followed by monocytes infiltration, stimulated by inflammatory mediators such as proinflammatory hormones, histamine, and cytokines, and which occurs immediately with the hypothermia and edema caused by thawing of the frozen tissue. Some scholars believe that inflammatory cell infiltration contributes to the development of apoptosis and tissue destruction [16]. As granulation tissue gradually forms, fibroblasts differentiate into myofibroblasts, and damaged collagen is replaced by new collagen. Cell infiltration helps to establish a new microcirculation system, which is essential for the repair process of tissues [17]. Delayed healing is one of the characteristics of cryogenic wounds, both in terms of scabbing and resorption, and the removal of necrotic tissue takes some time. Frozen wounds heal more slowly than excisional wounds, but the microcirculatory system is improved.

4 Current status of cryotherapy

4.1 Cryotherapy of skin diseases. Cryotherapy of dermatosis has been relatively common, with high cure rate and recurrence rate of about 10%, as shown in Table 1[18]. In the treatment of small basal and squamous cell carcinomas (2 cm in diameter), Kuflik[19] in his summary of 30 years of clinical experience with 4,406 skin cancers found a 98% cure rate with cryotherapy. A study by Nordin and Stenquist[20] on the outcome of 100 auricular cryosurgical treatments over a 5-year period found only one recurrence and concluded that surgical resection followed by cryotherapy of the lesion is a safe treatment.

Table 1 Freezing Time and Treatment Results for Benign, Precancerous Dermatosis

4.2 Cryotherapy of malignant tumors. Most tumors are best treated by surgical resection, which facilitates surgical examination and evaluation. Conservative surgical treatment is minimally invasive and ablative treatment (such as cryosurgery). Cryosurgery is a safe and effective treatment for a variety of tumors, mainly applied to a variety of solid tumors in the whole body, including liver cancer, lung cancer, prostate cancer, colorectal cancer, glioma, renal cancer, benign and malignant tumors of the bone, melanoma, breast cancer, pancreatic cancer, soft-tissue tumors, adrenal carcinomas, meningiomas, and uterine leiomyomas, as well as pain relief for cancers, which is now most commonly used for liver cancer and lung cancer [21-36]. Cancerous skin is mainly treated with hand-held liquid nitrogen spray device. See Table 2 for cryotherapy time and treatment results [37]. In vivo tumor cryotherapy is mainly performed using the argon-helium knife and the Combo knife. Since the introduction of argon-helium knife in China in 1999, more than 20,000 tumor patients have received argon-helium knife cryoablation, with an effective rate of more than 90%[38]. Cryosurgery is effective, but not optimal. Studies have shown that argon-helium knife cryoablation combined with chemotherapy drugs or multiple immunotherapies can significantly improve the survival rate of patients [39-40]. The study by Liu Bing and Zhang Yuanhao [41] showed that the liver metastases of colorectal cancer treated with argon-helium knife cryoablation combined with autologous cellular immunotherapy were more significantly reduced than those treated with cryotherapy alone and autologous cellular immunotherapy alone. A study by Liang et al [42] showed that the combination of cryoablation, natural killer cell therapy and Herceptin in the treatment of recurrent breast cancer resulted in better reduction of circulating tumor cell levels, reduction of carcinoembryonic antigen and glycoantigens15-3, and significant enhancement of immune function, compared to either the cryoablation alone or the combination of cryoablation and natural killer cell therapy. The Combo knife pioneered in China has completed clinical trials and can be widely used in cryotherapy of clinical solid tumors. The first clinical application was carried out in Peking University Cancer Hospital on July 13, 2018. At present, it has not been combined with other therapies and drugs [43].

Table 2 Freezing Time and Results of Cryotherapy for Cancerous Skin

AIDS: acquired immune deficiency syndrome

5. Summary

Cryotherapy has a wide range of applications and high clinical value. At present, the cooling rate, mechanism and therapeutic effect of indications of cryotherapy have been fully studied, especially for argon-helium knife cryoablation, but the clinical study on Combo knife with short application time is not sufficient. Combo knife has the advantages of low price, low operation cost and overall performance superior to argon-helium knife, which is more suitable for clinical application in China. It is very important to determine the treatment scheme of argon-helium knife or Combo knife combined with multiple immunotherapies, the type and delivery mode of combined drugs, as well as the freezing time, rewarming time and time interval between freeze-thaw cycles, for improving the clinical cryotherapy effect, but further study is still needed. In addition, the problem of immune tolerance caused by the release of tissue antigens by cell destruction after cryosurgery also needs to be addressed. The further study of cryotherapy immune response, mainly the changes of cytokine release and T cell differentiation on gene expression and related signal pathways, is of great significance to the understanding of the essence of cryotherapy.

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Received on: October 24, 2018 Revised on: 2018-11-20 Editor: Li Jin